KR20220170253A - Lactobacillus fermentum JNU532 strain and composition for skin whitening comprising cell-free supernatant thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus fermentum JNU532 strain and a skin whitening composition comprising cell-free supernatant thereof. The Lactobacillus fermentum JNU532 strain with accession number KCCM12987P exhibits excellent antioxidant activity, melanogenesis inhibitory activity, acid resistance and bile acid resistance.

Description

Lactobacillus fermentum JNU532 strain and composition for skin whitening comprising cell-free supernatant thereof

The present invention relates to Lactobacillus fermentum JNU532 strain.

Lactic acid bacteria are useful microorganisms used in the manufacture of fermented foods such as yogurt and kimchi, medicines, and feed additives (probiotics). . Lactic acid bacteria are anaerobic bacteria that do not produce catalase, but have a mechanism to remove active oxygen to protect cells from active oxygen generated during metabolism. It has been found that the antioxidant action of lactic acid bacteria is mainly due to metal ion chelation, active oxygen scavenging action, and reduction action. The Lactobacillus genus, which is widely used as probiotics, has recently been reclassified into several genera in the microbiological world, and Lactobacillus fermentum has been reclassified as Limosilactobacillus fermentum . However, since the use of the new genus name has not yet been expanded, the genus name of Lactobacillus was used in the present invention.

Melanin is a skin pigment produced by melanocytes through a metabolic pathway known as melanogenesis, and the change in skin color is due to increased synthesis of melanin for the purpose of suppressing damage to skin cells caused by UV absorption. The enzyme that plays the most important role in melanin production is tyrosinase, which oxidizes DOPA with tyrosine hydroxylase that oxidizes tyrosine in the melanosome to produce 3,4-dihydroxyphenylalanine (DOPA). It acts as DOPA oxidase to make dopachrome and finally produces melanin polymer. The antioxidant activity of lactic acid bacteria can ultimately prevent skin pigmentation by inhibiting melanin synthesis in the melanogenesis pathway by inactivating active oxygen that affects melanocytes.

Korea Patent No. 1937130

An object of the present invention is to provide a strain of Lactobacillus fermentum JNU532.

An object of the present invention is to provide a cosmetic composition for antioxidant or whitening.

An object of the present invention is to provide a food composition for antioxidant or whitening.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating pigmentary diseases caused by hyperpigmentation.

1. Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain.

2. The strain according to 1 above, wherein the strain has a 16S rDNA sequence represented by SEQ ID NO: 1.

3. The method of 1 above, wherein the strain has tyrosinase activity inhibition ability, TYR (tyrosinase) gene expression inhibition ability, TRP-1 (tyrosinase related protein-1) gene expression inhibition ability, TRP-2 (tyrosinase related protein-2) ) A strain having gene expression suppression ability and MITF (microphthalmia-associated transcription factor) gene expression suppression ability.

4. Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain, antioxidant or whitening cosmetic composition comprising any one selected from the group consisting of a culture medium of the strain and a cell-free supernatant of the strain.

5. Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain, antioxidant or whitening food composition comprising any one selected from the group consisting of a culture medium of the strain and a cell-free supernatant of the strain.

6. Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (Accession No. KCCM12987P) strain, culture of the strain, and prevention of pigmentary diseases caused by hyperpigmentation, including any one selected from the group consisting of cell-free supernatant of the strain, or Therapeutic pharmaceutical composition.

7. The pharmaceutical composition according to 6 above, wherein the pigment disease is any one selected from the group consisting of: melasma, freckles, senile pigment spots, blemishes, birthmarks, solar lentigines, and melanoma.

The Lactobacillus fermentum JNU532 strain of the present invention, a culture solution of the strain, or a cell-free supernatant of the strain has excellent antioxidant activity and melanin production inhibitory activity, and thus can help in skin whitening. In addition, the present invention Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 strain has excellent acid resistance and bile acid resistance, so it is possible to reach the intestine without dying when passing through the stomach.

1 is a result of confirming the degree of antioxidant activity of 19 Lactobacillus strains.
Figure 2 is the result of confirming the degree of melanin production inhibitory activity of five Lactobacillus strains.
Figure 3 is the result of confirming the cytotoxicity and melanin content of B16F10 cells treated with cell-free supernatants of five Lactobacillus strains.
Figure 4 is the result of confirming the melanogenesis-related gene and protein inhibitory effect of the cell-free supernatant of Lactobacillus fermentum JNU532.
5 is a result of confirming the acid resistance and bile acid resistance of Lactobacillus fermentum JNU532.

Hereinafter, the present invention will be described in detail.

The present invention provides a Lactobacillus fermentum JNU532 strain.

The genus Lactobacillus has recently been reclassified into several genera in microbiology, and Lactobacillus fermentum has been reclassified as Limosilactobacillus fermentum . However, since the use of the new genus name has not yet been expanded, the genus name of Lactobacillus was used in the description of the present invention.

Lactobacillus fermentum JNU532 strain has a 16S rDNA sequence represented by SEQ ID NO: 1.

The Lactobacillus fermentum JNU532 strain was deposited with the Korean Culture Center of Microorganisms and registered under accession number KCCM12987P.

Lactobacillus fermentum JNU532 strain may be isolated from kimchi.

Lactobacillus fermentum JNU532 strain exhibits excellent antioxidant activity. Specifically, the Lactobacillus fermentum JNU532 strain has excellent DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) radical scavenging activity and It has FRAP (ferric reducing antioxidant power) reducing ability.

Lactobacillus fermentum JNU532 strain exhibits excellent melanogenesis inhibitory activity. Specifically, the Lactobacillus Fermentum JNU532 strain has excellent tyrosinase activity inhibition ability, TYR (tyrosinase) gene, TRP-1 (tyrosinase related protein-1) gene, TRP-2 (tyrosinase related protein-2) gene and It has the ability to suppress MITF (microphthalmia-associated transcription factor) gene expression.

The Lactobacillus fermentum JNU532 strain exhibits acid resistance and bile acid tolerance. Therefore, it is possible to reach the intestine without being killed when passing through the stomach.

The present invention provides a cosmetic composition for antioxidant or whitening.

The cosmetic composition for antioxidant or whitening includes any one selected from the group consisting of a Lactobacillus fermentum JNU532 strain, a culture medium of the strain, and a cell-free supernatant of the strain.

The Lactobacillus fermentum JNU532 strain may be within the above range, but is not limited thereto.

The Lactobacillus fermentum JNU532 strain, the culture medium of the strain, and the cell-free supernatant of the strain show excellent antioxidant activity and melanogenesis inhibitory activity, and thus can be used as antioxidant or whitening cosmetic compositions.

The culture medium refers to the culture medium itself in which the strain is cultured.

The cell-free supernatant refers to a filtrate in which the strain is removed by filtering or centrifuging the culture medium in which the strain is cultured.

Antioxidation is an action of scavenging active oxygen, and typical factors for increasing active oxygen include stress, ultraviolet rays, radiation, and the like.

Whitening refers to any action that suppresses or prevents skin deposition by inhibiting the synthesis of melanin.

The cosmetic composition for antioxidant or whitening of the present invention may include ingredients commonly used in cosmetic compositions in addition to active ingredients, such as antioxidants, stabilizers, solubilizers, conventional adjuvants such as vitamins, pigments and fragrances, and carriers. includes

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , It may be formulated into oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, removed, or washed with water. As specific examples, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel, and a cleansing pack, and the surfactant-free cleansing formulation is a cleansing cream. , cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

The present invention provides a food composition for antioxidant or whitening.

The antioxidant or whitening food composition includes any one selected from the group consisting of Lactobacillus fermentum JNU532 strain, a culture medium of the strain, and a cell-free supernatant of the strain.

Lactobacillus fermentum JNU532 strain, culture medium, cell-free supernatant, antioxidant and whitening may be within the above range, but are not limited thereto.

The Lactobacillus fermentum JNU532 strain, the culture medium of the strain, and the cell-free supernatant of the strain show excellent antioxidant activity and melanogenesis inhibitory activity, and thus can be used as antioxidant or whitening food compositions. In addition, the strain exhibits acid resistance and bile salt tolerance, so it is not killed when passing through the stomach and can reach the intestine, which is advantageous when used as a food composition.

The food composition may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of antioxidant or whitening.

The food composition of the present invention may contain conventional food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. and judged by criteria.

Items listed in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, alkali additives for noodles, preservative preparations, and tar color preparations.

For example, a food composition in the form of a tablet is obtained by granulating a mixture obtained by mixing the composition with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding or directly compressing the mixture. can be molded. In addition, the food composition in the form of a tablet may contain a flavoring agent and the like, if necessary.

Among food compositions in the form of capsules, hard capsules can be prepared by filling a mixture obtained by mixing the composition with additives such as excipients in a conventional hard capsule, and soft capsules are a mixture obtained by mixing the composition with additives such as excipients. It can be prepared by filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like, if necessary.

The food composition in the form of a ring may be prepared by molding a mixture obtained by mixing the composition with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and, if necessary, may be coated with sucrose or other coating agent, or starch, The surface can also be coated with a material such as talc.

A food composition in the form of a granule may be prepared by a conventionally known method of a mixture of the composition and an excipient, a binder, a disintegrant, etc., and may contain a flavoring agent, a flavoring agent, and the like, if necessary.

Food compositions include beverages, meat, chocolate, foods, and confectionery. It may be pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

The food composition may be applied orally for use as a nutrient, and the application form is not particularly limited. For example, when administered orally, the daily intake is preferably 5000 mg or less, more preferably 2000 mg or less, and most preferably 500 to 1500 mg or 650 mg daily. When formulated into capsules or tablets, one tablet can be administered with water once a day.

The present invention provides a pharmaceutical composition for preventing or treating pigmentary diseases caused by hyperpigmentation.

The pharmaceutical composition for preventing or treating pigmentary diseases caused by hyperpigmentation includes any one selected from the group consisting of a Lactobacillus fermentum JNU532 strain, a culture medium of the strain, and a cell-free supernatant of the strain.

The Lactobacillus fermentum JNU532 strain, the culture medium, and the cell-free supernatant may be within the above range, but are not limited thereto.

The Lactobacillus fermentum JNU532 strain, the culture medium of the strain, and the cell-free supernatant of the strain exhibit excellent antioxidant activity and melanin production inhibitory activity, and thus can be used as a pharmaceutical composition for preventing or treating pigmentary diseases caused by hyperpigmentation.

Pigmentation diseases caused by hyperpigmentation can be caused by the excessive production and distribution of melanin in the epidermis, which is synthesized in melanosomes containing tyrosinase, the main pigment enzyme in mammalian melanocytes. .

Pigmented diseases due to hyperpigmentation are not limited as long as they are caused by or affected by excessive synthesis of melanin, and include, for example, melasma, freckles, senile pigment spots, blemishes, birthmarks, solar lentigines and melasma. It may be any one selected from the group consisting of melanoma.

The term “prevention” refers to prophylactic measures that result in any degree of reduction in the likelihood of developing a condition to be prevented or a recurrence or recurrent condition, including minor, substantial or major reduction in the likelihood of developing or recurring the condition, as well as total prevention. Refers to a measure, and the degree of likelihood reduction is at least a minor reduction.

The term "treatment" refers to treatment that results in a beneficial effect on a subject or patient suffering from the condition being treated, including not only cure but also relief of any degree, including minor, substantial, major relief, the degree of relief being At least a slight relief.

The pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. It can, but is not limited thereto.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc., such solid preparations may contain at least one excipient such as starch or calcium carbonate in the compound. It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

In the pharmaceutical composition of the present invention, the effective amount may vary depending on the patient's age, sex, and body weight, and is generally 1 to 6000 mg per kg of body weight, preferably 60 to 600 mg may be administered once or divided into three times. there is. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

Hereinafter, examples will be described in detail to explain the present invention in detail.

Example

Materials and Methods

1. Sample preparation

Nineteen Lactobacillus strains (Table 1) were cultured for 24 hours at 37° C. in MRS medium (BD, USA). The growth rates of the 19 Lactobacillus strains over 24 hours were different. Therefore, in order to integrate the number of Lactobacillus strains, the absorbance value at 600 nm of the suspension of 19 Lactobacillus strains was measured, and the suspension was adjusted to 10 ⁸ cells/mL at the same concentration. Each suspension was centrifuged (3,500 xg, 4°C) for 15 minutes to obtain a cell-free supernatant (CFS, cell-free supernatant). CFS was filtered using a 0.2 micrometer syringe (Sartorius AG, Germany) and stored at -20°C.

number. strain name abbreviation separation circle Remarks (Accession No.) One Lactobacillus curvatus BYB1 BYB1 kimchi - 2 Lactobacillus curvatus BYB2 BYB2 kimchi - 3 Lactobacillus curvatus BYB3 BYB3 kimchi - 4 Lactobacillus curvatus BYB4 BYB4 kimchi - 5 Lactobacillus curvatus BYB7 BYB7 kimchi - 6 Lactobacillus brevis OB1 OB1 kimchi - 7 Lactobacillus brevis OB4 OB4 kimchi - 8 Lactobacillus brevis OB3 OB3 kimchi - 9 Lactobacillus sakei OB8 OB8 kimchi - 10 Lactobacillus casei MYA5 MYA5 kimchi - 11 Lactobacillus sakei JNU830 JNU830 kimchi KCCM11586P 12 Lactobacillus sakei MYA6 MYA6 kimchi - 13 Lactobacillus amylovorus KCNU KCNU small intestine KCCM-10753P 14 Lactobacillus acidophilus GP1B GP1B small intestine - 15 Lactobacillus plantarum L67 L67 infant feces KCCM11522P 16 Lactobacillus plantarum OY1 OY1 kimchi - 17 Lactobacillus plantarum OY2 OY2 kimchi - 18 Lactobacillus fermentum JNU532 JNU532 kimchi - 19 Lactobacillus fermentum JNU534 JNU534 kimchi KCCM129879P * 24-hour stationary culture at 37 ° C using MRS medium

2. Cell culture

B16F10 cells (ATCC®CRL-6475™) were cultured in 10% fetal bovine serum (FBS) (Gibco, USA) and 1% antibiotic-antimycotic under conditions containing 5% CO ₂ at 37°C. ) in Dulbecco's modified Eagle's medium (DMEM)/high-glucose medium (Hyclone, USA). Medium was changed three times a week. Cells were seeded in 96-well or 6-well flat-bottomed plates. The final volume was 100 μL/well in a 96-well culture plate and 1 mL/well in a 6-well culture plate. After culturing for 24 hours, cells were separated using 0.05% Trypsin-ethylenediaminetetraacetic acid (Gibco, USA) and separated for 5 minutes by centrifugation (1000 xg, 4°C). The supernatant was discarded and the cell pellet was washed twice with cold phosphate buffer (pH=7.4).

3. Evaluation of antioxidant activity

Antioxidant activity of Lactobacillus strains was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging assay and ferric reducing antioxidant power. (FRAP). The DPPH scavenging activity method developed by Blois (1960) was used with several modifications. Briefly, 150 μL of a 0.1 mM DPPH (Sigma-Aldrich, USA) solution prepared in methanol and 50 μL of CFS of each Lactobacillus strain were added to a 96-well microplate. Control wells contained only methanol and 0.05 mg/mL ascorbic acid was added as a positive control. The reaction mixture was incubated in the dark at 25° C. for 30 minutes, and absorbance at 517 nm was measured using a microplate reader (Synergy HTX; Biotek, USA). DPPH scavenging activity was calculated using Equation 1 below.

[Equation 1]

DPPH scavenging activity (%) = [(Absorbance of ascorbic acid - Absorbance of sample)/Absorbance of ascorbic acid] × 100%

ABTS clearance analysis was performed using ABTS (Sigma-Aldrich) according to the developer's instructions. ABTS is oxidized to green ABTS+• by the action of an oxidizing agent, and the production of ABTS+• is suppressed in the presence of an antioxidant. The ABTS working solution consisted of 7.4 mM ABTS solution and 2.6 mM potassium persulfate diluted in phosphate buffered saline (PBS, pH=7.4) before performing the assay. For the ABTS clearance assay, 150 μL of ABTS working solution and 50 μL of Lactobacillus CFS were added to a 96-well microplate. Control wells contained only PBS and 0.05 mg/mL ascorbic acid was added as a positive control. The reaction mixture was incubated at 25° C. for 30 minutes in the dark and absorbance was measured at 734 nm using a microplate reader (Synergy HTX). ABTS scavenging activity was calculated using Equation 2.

[Equation 2]

ABTS scavenging activity (%) = [(Absorbance of ascorbic acid - Absorbance of sample)/Absorbance of ascorbic acid] × 100%

FRAP reducing capacity evaluation to determine total antioxidant capacity is based on the principle that antioxidants can reduce blue ferrous tripyridyl triazine (Fe ²⁺ -TPTZ) produced by ferric tripyridyl triazine (Fe ³⁺ )-TPTZ under acidic conditions. do. The total antioxidant capacity of a sample can be determined by measuring the content of Fe ²⁺ -TPTZ. For the FRAP assay, the FRAP working solution was 50 mL of 300 mM acetate buffer (pH=3.6), 5 mL of 10 mM TPTZ solution (Sigma-Aldrich) and 5 mL of 20 mM iron (III) chloride hexahydrate. (FeCl ₃ .6H ₂ O) was freshly prepared by mixing (Sigma-Aldrich). The reaction mixture consisted of 150 μL of FRAP working solution and 50 μL of Lactobacillus CFS in a 96-well microplate, and 0.05 mg/mL ascorbic acid was used as a positive control. The reaction mixture was incubated at room temperature for 30 minutes in the dark, and absorbance was measured at 593 nm using a microplate reader (Synergy HTX). A FRAP standard curve was generated using 1 mM iron (II) sulfate hexahydrate (FeSO ₄ 7H ₂ O), and standard solutions at concentrations of 0, 20, 40, 60, 80, and 100 μM were prepared. FRAP values were calculated using Equation 3.

[Equation 3]

Ascorbic Acid Equivalent FRAP value (%) = (Absorbance of sample/Absorbance of 0.05 mg/mL ascorbic acid) × 100%

4. Cell-free tyrosinase assay

L-tyrosine was used as a reaction substrate in the tyrosine activity assay. Briefly, 100 μL of 200 units/mL mushroom tyrosinase (Sigma-Aldrich) prepared in phosphate buffer (pH=6.5), 50 μL of 1 mM L-tyrosinase, 10 μL of CFS of Lactobacillus and 40 μL of distilled water (dH ₂ O) was added to a 96-well microplate; dH ₂ O was used as a negative control and 500 μM arbutin (Sigma-Aldrich) was used as a positive control. After measuring the initial absorbance at 490 nm using a microplate reader (Synergy HTX), the plate was incubated at 37°C for 30 minutes, and then the absorbance was measured again. Cell-free tyrosinase activity was calculated using Equation 4.

[Equation 4]

Inhibition of tyrosinase activity (%) = [(A - B) (C - D)]/ (A - B) × 100%

(Wherein, A is the final absorbance of the control group, B is the initial absorbance of the control group, C is the final absorbance of the sample, and D is the initial absorbance of the sample)

5. Cell viability assay

The viability of melanoma cells was determined by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B16F10 cells (1×10 ³ cells/mL) treated with serum-free DMEM containing 10% Lactobacillus CFS were cultured at 37° C. in 5% CO ₂ humidified air for 24 hours. 100 microliters of the cultured mixture was replaced with a 5 mg/mL MTT solution (Sigma-Aldrich) dissolved in phosphate buffer (pH=6.8) and incubated again for 4 hours. The MTT solution was removed and 100 μL of dimethyl sulfoxide (Sigma-Aldrich) was added. Absorbance was measured at 490 nm using a microplate reader (Synergy HTX).

6. Measurement of melanin content

B16F10 cells (1x10 ⁵ cells/mL) were treated with serum-free DMEM containing 10% Lactobacillus CFS. After culturing for 24 hours, the medium was removed and the cells were collected. To measure melanin content, cells were lysed in 1N NaOH at 70°C for 1 hour. Cells were grown in DMEM without treatment with Lactobacillus CFS as a control. MRS medium (pH=6.5, pH=4, pH=3) was used as a negative control. Absorbance was measured at 490 nm using a microplate reader (Synergy HTX).

7. Intracellular tyrosinase activity assay

B16F10 cells (1x10 ⁵ cells/mL) were treated with serum-free DMEM containing 10% Lactobacillus CFS for 24 hours. B16F10 cells were collected and lysed at -80°C for 1 hour using phosphate buffer (pH=6.8) containing 1% Triton X-100 (Thermo Fisher Scientific, USA). After lysis, the cells were centrifuged at 14,000 xg at 4°C for 10 minutes to collect the supernatant. Supernatant (90 μL of cell lysate) and 10 μL of 2 mg/mL L-3,4-dihydroxyphenylalanine (Sigma-Aldrich) were added to a 96-well microplate and incubated at 37°C for 30 minutes. Cells grown in DMEM without Lactobacillus CFS were used as controls. Absorbance was measured at 490 nm using a microplate reader (Synergy HTX). Inhibition of tyrosinase activity was calculated using Equation 5.

[Equation 5]

Inhibition of tyrosinase activity (%) = (1 - Absorbance of sample/Absorbance of control) × 100%

8. Quantitative reverse-transcription polymerase chain reaction (RT-PCR)

B16F10 cells (1x10 ⁵ cells/mL) treated with serum-free DMEM containing 10% Lactobacillus CFS were cultured for 24 hours. B16F10 cells were harvested and total cellular RNA was extracted using the Pure Helix™ Total RNA Purification Kit (Nano Helix, South Korea) according to the manufacturer's instructions, and the RNA was stored at -70°C. The RNA concentration of the sample was diluted to 0.05 mg/μL using dH ₂ O. Template RNA (20 μL) was added to Maxime RT-PCR PreMix tubes (iNtRON Biotechnology, South Korea). Complementary DNA (cDNA) was synthesized as follows: cDNA synthesis at 5°C for 60 min, reverse transcriptase inactivation step at 95°C for 5 min, diluted reaction to 50 μL with sterile water, stored at -20°C. cDNA was synthesized using the KAPA SYBR®FAST qPCR Master Mix (2X) kit (KAPA Biosystems, South Africa) according to the manufacturer's instructions. The PCR cycle was as follows: denaturation at 95°C for 30 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 30 seconds. Each PCR reaction was performed for 35 cycles for semi-quantitative evaluation of mRNA levels. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) primer was used as a control. The RT-PCR primer sequences used are listed in Table 2.

Gene Primer sequences (5'-3') sequence number GADPH Forward TCACCACCATGGAGAAGGC 2 Reverse GCTAAGCAGTTGGTGGTGCA 3 TYR
(tyrosinase) Forward ACACCTGAGGGGACCACTAT 4 Reverse CATTGGCTTCTGGGTAAACT 5 TRP-1
(tyrosinase related protein-1) Forward GCCACAAGGAGGTTAGAAGACA 6 Reverse CCAGTAAGGAAGGGAGAAAGAG 7 TRP-2
(tyrosinase related protein-2) Forward AGAAGTTTGACAGCCCTCC 8 Reverse CAAGTTGCTCTGCGGTTAG 9 MITF
(microphthalmia-associated transcription factor) Forward AACGGGAACAGCAACGAGC 10 Reverse TCACCAGATCAGGCGAGCA 11

9. Western blot analysis

B16F10 cells (1x10 ⁵ cells/mL) treated with serum-free DMEM containing 10% Lactobacillus CFS were cultured for 24 hours. B16F10 cells were collected by centrifuging this mixture at 3000 xg, 4°C for 5 minutes. After obtaining the cell pellet, the total protein of the cells was extracted using PRO-PREP™ protein extraction solution (iNtRON Biotechnology), and the total protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Sample proteins were mixed with sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris-HCl, 400 mM dithiothreitol, 8% SDS, 6 mM bromophenol blue, 4.3 M glycerol), denatured at 95 °C for 5 min, and then incubated on ice for 5 min. left for a minute. Sample proteins (15 μL) were added to polyacrylamide gel wells and separated using 8% SDS-polyacrylamide gel electrophoresis. The gel was transferred to polyvinylidene difluoride membranes (Bio-Rad, USA) and blocked for 60 min at room temperature in 5% skimmed milk powder prepared with 0.1% Tween 20 in PBS. Washed twice with 0.05% PBST (0.05% Tween 20 in PBS). Membranes were incubated with mouse monoclonal antibodies: β-actin (C4, 1:2000 dilution), TYR (T311, 1:100 dilution), TRP-1 (G-9, 1:100 dilution), TRP-2 (C -9, 1:100 dilution) and MITF (C5, 1:100 dilution) antibodies (diluted in 5% skim milk powder in 0.1% PBST) were diluted at room temperature for 2 hours or at 4°C overnight. The membrane was washed three times for 10 minutes each time in 0.05% PBST, followed by incubation for 1 hour 30 minutes at room temperature with anti-mouse (diluted 1:5000) secondary antibody (diluted using 5% skim milk powder in 0.1% PBST). Then, it was washed again 3 times for 10 minutes with 0.05% PBST. Western blotting detection kit (ELPISBIO, Korea) was used according to the manufacturer's instructions. Briefly, the membrane was placed in a container and 0.5 mL of solution A and 0.5 mL of solution B were added to completely cover the membrane before incubating for 1 min. Specific proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, US). β-actin expression was used as an internal control to demonstrate equal loading of protein samples.

10. Acid resistance and bile acid tolerance

To evaluate the acid resistance of the Lactobacillus strain used in the present invention, 100 μL of pepsin solution (1000 units/mL; Sigma-Aldrich) was added to sterile MRS medium (10mL) adjusted to pH 2.5, and then the Lactobacillus strain was inoculated at 37 ° C. cultured in. Acid resistance was evaluated by examining the difference between the number of viable cells after 1 hour and 2 hours of culture compared to the number of viable cells initially inoculated. Bile salt tolerance was calculated according to the method described by Gilliland et al. Lactobacillus was inoculated into MRS medium supplemented with 0.3% bile (oxgall), and bile acid resistance was evaluated by examining the number of viable cells after 24 hours and 48 hours of incubation compared to the number of viable cells at the beginning of inoculation at 37 ° C.

11, statistical analysis

Results were analyzed using one-way analysis of variance and expressed as mean ± standard deviation. Analysis was performed using IBM SPSS for Windows Ver. 23 (SPSS, Chicago, IL, USA), and values of ρ<0.05(*), ρ<0.01(**) and ρ<0.001(***) were considered significant.

result

1. Antioxidant activity of cell-free supernatants of Lactobacillus species

The identified Lactobacillus species are listed in Table 1 above. Lactobacillus curvatus BYB3, L. curvatus BYB4, L. brevis OB3, L. sakei OB8, L. casei MYA5, L. sakei JNU830, L. sakei MYA6, and L. fermentum JNU532 exhibited about 60% DPPH radical scavenging activity. Lactobacillus brevis OB1, L. brevis OB3, L. sakei OB8, and L. sakei MYA6 exhibited 30% ABTS+· scavenging activity (FIG. 1). Lactobacillus curvatus BYB7, L. brevis OB3, L. sakei OB8, L. casei MYA5, L. sakei JNU830 and L. fermentum JNU532 showed ascorbic acid equivalent FRAP values greater than 40% (FIG. 1). ). Lactobacillus sakei OB8, L. casei MYA5, L. sakei JNU830, L. sakei MYA6, and L. fermentum JNU532 were screened and evaluated for their antimelanogenic activity.

2. Tyrosinase activity of cell-free supernatant of Lactobacillus species and intracellular tyrosinase activity of B16F10 cells treated with cell-free supernatant of Lactobacillus species

MRS (control) showed 38% of tyrosinase inhibitory activity. Lactobacillus sakei JNU830, L. sakei MYA6 and L. fermentum JNU532 showed tyrosinase inhibitory activity of 40%; L. sakei OB8 and L. casei MYA5 showed less than 30% tyrosinase inhibitory activity. This suggests that the lactic acid culture supernatant sample cannot inhibit tyrosinase activity to reduce melanin content. DMEM control showed 100% tyrosinase activity, and arbutin inhibited 14% of tyrosinase activity. Lactobacillus sakei OB8 and L. casei MYA5 showed less than 5% tyrosinase inhibitory activity. Lactobacillus sakei JNU830, L. sakei MYA6 and L. fermentum JNU532 exhibited tyrosinase inhibitory activity of more than 15% (FIG. 2). Considering the melanin content and intracellular tyrosinase activity, L. fermentum JNU532, which has a strong antimelanogenic potential, was selected for further experiments. Subsequently, qRT-PCR and Western blotting were performed to investigate the melanogenesis inhibitory activity mechanism of L. fermentum JNU532.

3. Cytotoxicity and melanin content of B16F10 cells treated with Lactobacillus cell-free supernatant

As a result of evaluation using DMEM as a control, cells treated with cell-free supernatant (CFS) of L. sakei OB8, L. casei MYA5, L. sakei JNU830 and L. fermentum JNU532 exhibited 100% viability. The viability of cells treated with CFS of L. sakei MYA5 was 84%. The viability of cells treated with 500 μM Arbutin and MRS was 111% and 123%, respectively. CFS of L. sakei OB8, L. casei MYA5, and L. sakei MYA6 inhibited melanin production by 15%, 11%, and 14%, respectively. CFS of L. sakei JNU830 and L. fermentum JNU532 inhibited melanin production by 21% and 23%, respectively (FIG. 3). The melanin content of MRS-treated B16F10 cells (pH=6.5, pH=4, pH=3) was the same as that of the control group. Therefore, it was assumed that MRS medium (pH=6.5, pH=4, pH=3) did not affect melanin production. Therefore, CFS instead of MRS was selected for further testing of the study.

4. L. fermentum In B16F10 cells in JNU532-derived cell-free supernatant TYR , TRP-1 , TRP-2 and MITF Inhibitory effect on gene and protein expression of

The expression of four genes was decreased in CFS-treated melanocytes (FIG. 4). Lactobacillus fermentum JNU532 CFS can reduce melanin content by suppressing the expression of key genes required for melanin synthesis, including tyrosinase family genes (especially TYR ). Western blotting analysis showed that CFS may have inhibited the transcription and expression of melanogenesis genes by inhibiting the expression of key proteins TYR, TRP-1 and TRP-2 and blocking the signals of the MITF transduction pathway.

5. L. fermentum Acid and bile acid tolerance of JNU532

The acid and bile acid tolerance results showed that L. fermentum JNU532 could survive for 2 hours in MRS medium (pH=2.5). In addition, L. fermentum JNU532 showed resistance to 0.3% ox gall bile salts (FIG. 5).

Korea Microbial Conservation Center (Overseas) KCCM12987P 20210507

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION CHONNAM NATIONAL UNIVERSITY <120> Lactobacillus fermentum JNU532 strain and composition for skin whitening comprising cell-free supernatant <130> 21P05007 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 1189 <212> DNA <213> Lactobacillus fermentum <400> 1 ggacgtcgtc tgctataatg cagtcgaacg cgttggccca attgattgat ggtgcttgca 60 cctgattgat tttggtcgcc aacgagtggc ggacgggtga gtaacacgta ggtaacctgc 120 ccagaagcgg gggacaacat ttggaaacag atgctaatac cgcataacaa cgttgttcgc 180 atgaacaacg cttaaaagat ggcttctcgc tatcacttct ggatggacct gcggtgcatt 240 agcttgttgg tggggtaacg gcctaccaag gcgatgatgc atagccgagt tgagagactg 300 atcggccaca atgggactga gacacggccc atactcctac gggaggcagc agtagggaat 360 cttccacaat gggcgcaagc ctgatggagc aacaccgcgt gagtgaagaa gggtttcggc 420 tcgtaaagct ctgttgttaa agaagaacac gtatgagagt aactgttcat acgttgacgg 480 tatttaacca gaaagtcacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540 aagcgttatc cggatttatt gggcgtaaag agagtgcagg cggttttcta agtctgatgt 600 gaaagccttc ggcttaaccg gagaagtgca tcggaaactg gataacttga gtgcagaaga 660 gggtagtgga actccatgg tagcggtgga atgcgtagat atatggaaga acaccagtgg 720 cgaaggcggc tacctggtct gcaactgacg ctgagactcg aaagcatggg tagcgaacag 780 gattagatac cctggtagtc catgccgtaa acgatgagtg ctaggtgttg gagggtttcc 840 gcccttcagt gccggagcta acgcattaag cactccgcct ggggaggtacg accgcaaggt 900 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960 agctacgcga agaaccttac caggtcttga catcttgcgc caaccctaga gataggcgtt 1020 tccttcggga acgcaatgac aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt 1080 tggttaagtc ccgcaacgag cgcaaccctt gtactagtgc agcattaagt tgggcactct 1140 agtggagact gccggtgaca accgggagga agggtggggg acgacgtcc 1189 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 2 tcaccaccat ggagaaggc 19 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 3 gctaagcagt tggtggtgca 20 <210> 4 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 4 acacctgagg gaccactat 19 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 5 cattggcttc tgggtaaact 20 <210> 6 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 6 gccacaagga ggttagaaga ca 22 <210> 7 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 7 ccagtaagga agggagaaag ag 22 <210> 8 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 8 agaagtttga cagccctcc 19 <210> 9 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 9 caagttgctc tgcggttag 19 <210> 10 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 10 aacgggaaca gcaacgagc 19 <210> 11 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 11 tcaccagatc aggcgagca 19

Claims (7)

Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain.
The strain according to claim 1, wherein the strain has a 16S rDNA sequence represented by SEQ ID NO: 1.
The method according to claim 1, wherein the strain is tyrosinase (tyrosinase) activity inhibition ability, TYR (tyrosinase) gene expression inhibition ability, TRP-1 (tyrosinase related protein-1) gene expression inhibition ability, TRP-2 (tyrosinase related protein-2) gene A strain having expression suppression ability and MITF (microphthalmia-associated transcription factor) gene expression suppression ability.
Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain, antioxidant or whitening cosmetic composition comprising any one selected from the group consisting of a culture medium of the strain and a cell-free supernatant of the strain.
Lactobacillus fermentum ( Lactobacillus fermentum ) JNU532 (accession number KCCM12987P) strain, antioxidant or whitening food composition comprising any one selected from the group consisting of a culture medium of the strain and a cell-free supernatant of the strain.
Lactobacillus fermentum JNU532 (accession number KCCM12987P) strain, for preventing or treating pigmentary diseases caused by hyperpigmentation, including any one selected from the group consisting of a culture solution of the strain and a cell-free supernatant of the strain pharmaceutical composition.
The pharmaceutical composition according to claim 6, wherein the pigment disease is any one selected from the group consisting of: melasma, freckles, senile pigment spots, blemishes, birthmarks, solar lentigines, and melanoma.

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